The ability to genetically manipulate hematopoietic cells has enabled the development of novel immunotherapies. In order to generalize immunotherapy to common cancers, such as ovarian, breast, and colon cancer, we have designed novel chimeric antibody/T-cell receptor genes which combine variable regions from mAb with T-cell signaling chains. We have demonstrated that the insertion of these chimeric receptor genes into T-cells allows them to recognize tumor antigens defined by the receptors. We are currently conducting a clinical trial for ovarian cancer patients based on this strategy. In addition, we have transduced hematopoietic stem cells with chimeric receptor genes, and have demonstrated that mice reconstituted with the gene-modified bone marrow cells exhibit decreased tumor growth when challenged with tumor cells expressing the corresponding antigens. Transducing hematopoietic stem cells with chimeric receptor genes may allow the continuous in vivo production of lymphocytes, macrophages, neutrophils, and NK cells directed against the tumor. In addition, we have designed chimeric receptors that recognize KDR, the receptor for VEGF that is overexpressed on tumor vasculature. This approach, which would be broadly applicable to many types of cancer, may allow us to target tumor vasculature using transduced T cells. Current efforts are focused on enhancing in vivo T-cell survival, proliferation, and migration to tumor. In order to improve in vivo proliferation of adoptively transferred T-cells, we have designed dual specific lymphocytes which recognize a strong immunogen, such as alloantigen, through the native T-cell receptor, and tumor through the chimeric receptor. In vivo stimulation of dual specific T-cells, which are both alloreactive and tumor reactive, with alloantigen results in a 10-fold increase in circulating T-cells compared to controls. We are currently testing dual specific T-cells in patients with advanced ovarian cancer. We are also performing studies using gene-modified dendritic cells, which are potent antigen presenting cells capable of activating quiescent lymphocytes. Thus, these cells may be instrumental in the in vivo generation of tumor reactive T cells. We have studied several methods to gene modify, activate, and induce the proliferation of dendritic cells in an effort to increase their effectiveness in stimulating an anti-tumor response. The stable introduction of antigen genes into dendritic cells may be a more effective antigen loading method compared to peptide pulsing, since the antigen would be constitutively expressed in vivo and could express multiple epitopes from a single gene. We have developed a technique to stably gene modify primary murine and human dendritic cells using retroviral vectors. Because retroviral transduction requires proliferating cells, we gene modified dividing hematopoietic progenitor cells followed by in vitro differentiation into dendritic cells. We have demonstrated that murine dendritic cells transduced with a model antigen gene can treat established pulmonary metastases. In addition, we have transduced human dendritic cells with the melanoma antigens MART-1 and GP100 and have demonstrated that these cells can be used to generate tumor-specific T-cells from peripheral blood lymphocytes. T-cell cultures generated from GP100 transduced DCs could recognize at least 3 distinct class I restricted epitopes from GP100, thereby demonstrating one of the advantages of gene transduction compared to pulsing with a single peptide epitope. We have also demonstrated that GP100 specific, class II restricted CD4+ T-cells could be generated using transduced DC. Most recently, we have demonstrated that lentiviral vectors can gene modify non-proliferating monocyte-derived dendritic cells.